Immunoassay reagents and method for determining cyclosporine

ABSTRACT

Cyclosporine derivatives useful as detectable tracer compounds for the immunoassay determination of cyclosporine are disclosed. The cyclosporine derivatives comprise a detectable moiety coupled to the amino acid at the first position (MeBmt) in cyclosporine, the second position (Abu) in cyclosporine, the third position (Sar) in cyclosporine, the eighth position (D-Ala) in cyclosporine, or the tenth position (MeLeu) in cyclosporine. A preferred cyclosporine derivative comprises a fluorescent moiety coupled to the hydroxyl group of the amino acid at the first position in cyclosporine, and is especially useful for the fluorescent polarization immunoassay determination of cyclosporine. A fluorescent polarization immunoassay method and test kit are also disclosed.

This application is a Divisional of U.S. Ser. No. 08/148,164 filed Nov.4, 1993, now U.S. Pat. No. 5,489,668, which is a Continuation of U.S.Ser. No. 07/952,488, filed Sep. 28, 1992 and now abandoned, which is aContinuation of U.S. Ser. No. 07/567,842, filed Aug. 15, 1990 and nowabandoned.

FIELD OF THE INVENTION

The present invention relates to reagents for determining the presenceor amount of cyclosporine in a test sample. In particular, the presentinvention relates to detectable tracer compounds for use inimmunoassays, especially fluorescent polarization immunoassays, fordetecting the presence or amount of cyclosporine and metabolites ofcyclosporine in a test sample.

BACKGROUND OF THE INVENTION

Cyclosporine is a cyclic undecapeptide of fungal origin (U.S. Pat. No.4,117,118; Ruegger, et al., Helvetica Chimica Acta, Vol. 59(4), pages1075-1092 (1976); U.S. Pat. No. 4,289,851; and Traber, et al., HelveticaChemica Acta, Vol. 60(4), pages 1247-1255 (1977) and Vol. 65(5), pages1655-1677 (1982)! which is commonly employed as a potentimmunosuppressive agent to prevent the rejection of transplanted organssuch as kidney, heart, bone marrow, and liver in humans. Theeffectiveness of cyclosporine has also been investigated in thetreatment of conditions such as psoriasis, conjunctivitis, arthritis,nephritis and autoimmune diseases Donnelly, et al., Therapeutic DrugMonitoring, Vol. 11(6), pages 696-700 (1989)!. While a certain level ofcyclosporine must be maintained in the bloodstream to prevent rejectionof transplanted organs, nephrotoxicity, hepatotoxicity, and other sideeffects can result from higher blood levels of the drug or fromprolonged exposure. Moreover, distribution and metabolism ofcyclosporine varies greatly between individuals as well as in a singleindividual during the course of therapy. Accordingly, it is necessary tomonitor the concentration or level of cyclosporine in biological fluidssuch as whole blood, plasma, and serum, for proper patient managementBurchart, et al., Drug Intelligence and Clinical Pharmacy, Vol. 20,pages 649-652 (1986) and Shaw, et al., Clinical Chemistry, Vol. 33(7),pages 1269-1288 (1987)!. Measurement of cyclosporine in blood, plasmaand serum has been complicated, however, by the presence of metabolitesof cyclosporine therein Maurer, et al., Drug Metabolism and Disposition,Vol. 12(10), pages 120-126 (1984)!, and the toxicities,immunosuppressive activities, and synergistic effects of thesemetabolites are being investigated Dindzans, et al., TransplantationProceedings, Vo. 19(4), pages 3490-3493 (1987); Yee, et. al.,Transplant. Proc., Volume 18, pages 774-776 (1986); and Ryffel, et. al.,Transplant. Proc., Volume 20 (supplement 2), pages 575-584 (1988)!.Although the measurement of cyclosporine independently from itsmetabolites is desirable, there is also the need for assays that measurethe metabolites as well as the parent drug (Donnelly, et al., supra).The metabolites of cyclosporine that have been identified in which thering is still intact result from the hydroxylations and demethylationsof the parent compound Maurer, et al., Drug Metabolism and Disposition,12(1), pages 120-126 (1984)!. The structures of cyclosporine and some ofits major metabolites are of the formula:

    __________________________________________________________________________     ##STR1##                                                                     METABOLITER.sub.1R.sub.2R.sub.3                                               __________________________________________________________________________     ##STR2##                                                                     __________________________________________________________________________     ##STR3##                                                                 

The structure of cyclosporine may be alternately represented by theformula: ##STR4## wherein "MeBmt" represents a residue ofN-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)-threonine; "MeVal"represents a residue of (N)-methyl-(L)-valine; "MeLeu" represents aresidue of (N)-methyl-L-leucine; "D-Ala" represents a residue ofD-alanine; "Ala" represents a residue of L-alanine; "Val" represents aresidue of L-valine; "Abu" represents a residue ofL-(alpha)-aminobutyric acid; and "Sar" represents a residue ofsarcosine, also known as N-methylglycine. The term "residue" refers tothe condensed form of the amino acid found in peptides, and theconfiguration of the alpha-amino acid is assumed to be L unless aD-configuration is specified. Conventional nomenclature for analogs ofcyclosporine are defined herein by reference to the structure ofcyclosporine (i.e., cyclosporin A) by first indicating those residues inthe molecule which differ from those present in cyclosporine, and thenapplying the term "cyclosporine" to characterize the remaining residueswhich are identical to those present in cyclosporine. Thus, Thr!²cyclosporine designates the cyclosporine in which the amino acid residuein the 2 position is threonine, i.e., cyclosporin C.

Cyclosporine levels in whole blood, plasma and serum have been measuredby high performance liquid chromatography (HPLC) Lensmeyer, et al.,Clinical Chemistry, Vol. 31(2), pages 196-201 (1985)!, radioimmunoassay(RIA) utilizing ³ H Donatsch et al., Journal of Immunoassay, Vol. 2(1),pages 19-32 (1981)! or ¹²⁵ I U.S. Pat. No. 4,727,035 and Mahoney, etal., Clinical Chemistry, Vol. 31(3), pages 459-462 (1985)!, fluorescentimmunoassays (U.S. Pat. No. 4,727,035), and by fluorescence polarizationimmunoassay (FPIA) Marty, et al., Analytical Letters, Vol. 22(13 & 14),pages 2717-2736 (1989) and European Patent Application Publication No.283,801!. While the metabolites of cyclosporine can be distinguishedfrom cyclosporine itself according to such HPLC methods, HPLC isnevertheless time and labor intensive, requiring extensive samplepreparation and at least thirty minutes to perform the assay. Similarly,RIA assays suffer from the disadvantages of using radioactive materialswhich require special storage, handling and disposal, and typicallyrequire a minimum of two hours to perform.

While fluorescent polarization immunoassays are superior to the methodsdescribed above, particularly in ease of use, commercially availablepolyclonal antibody immunoassays display a lack of specificity forcyclosporine over its metabolites. In this regard, the specificity ofimmunoassays is dependent upon the antibody used, and the relativeaffinities of the antibody for cyclosporine, metabolites ofcyclosporine, and the labeled form of cyclosporine. Recently, monoclonalantibodies specific for cyclosporine over its metabolites have beendescribed Quesniaux, et al., Immunology Letters, Vol. 12(1), pages120-126 (1985), Clinical Chemistry, Vol. 33(1), pages 32-37 (1987), andMolecular Immunology, Vol. 24(11), pages 1159-1168 (1987)!, and RIAassays using these antibodies have been found to correlate well withHPLC.

The present invention overcomes the disadvantages of the HPLC and RIAmethods described above by providing reagents which are particularlyuseful in immunoassays, especially fluorescent polarizationimmunoassays, for detecting cyclosporine or cyclosporine and metabolitesof cyclosporine. Moreover, the present invention is an advance overimmunoassays for cyclosporine which have been previously described byproviding novel tracer compounds for use in immunoassays, particularlyfluorescent polarization immunoassays, employing either specific ornonspecific antibodies to detect cyclosporine or cyclosporine andmetabolites of cyclosporine in a test sample.

SUMMARY OF THE INVENTION

The present invention relates to novel cyclosporine derivative compoundscomprising cyclosporine, or analogs of cyclosporine, labeled with adetectable moiety for use as a tracer compound in an immunoassay fordetermining the presence or amount of cyclosporine, or cyclosporine andmetabolites thereof, in a test sample. Preferably, the detectable moietyis fluorescein, or a derivative of fluorescein, wherein such fluorescenttracer compounds are particularly useful for performing fluorescentpolarization immunoassays.

The cyclosporine derivatives of the present invention comprise adetectable moiety coupled to cyclosporine, or derivatives ofcyclosporine, at the amino acids found at the first position(N-methyl(4R)-4-but-2E-en-1-yl-methyl-L-threonine residue) incyclosporine, the second position (L-alpha-aminobutyric acid residue) ofcyclosporine, the third position (N-methylglycine residue) ofcyclosporine, the eighth position 8 (D-alanine residue) of cyclosporine,or the tenth position (N-methyl-L-leucine residue) of cyclosporine,preferably to the hydroxyl group of the amino acid at the secondposition of cyclosporine. The cyclosporine derivatives of the presentinvention are represented by the formula: ##STR5## where R₁ is an aminoacid residue of the structure ##STR6## R₂ is an amino acid residue ofthe structure ##STR7## R₃ is an amino acid residue of the structure##STR8## R₄ is an amino acid residue of the structure ##STR9## R₅ is anamino acid residue of the structure ##STR10## wherein Z is a detectablemoiety capable of producing a detectable signal, with the proviso that:

when R₁ is so defined, X₁ is a linking group of 1-20 atoms excludinghydrogen, R₂ is a residue of (L)-alpha-aminobutyric acid or a residue of(L)-threonine, or a residue of (L)-threonine in which the hydroxyl groupis acylated with an acyl group of 1-10 atoms or a fluoresceinyl group;R₃ is a residue of sarcosine, or a residue of (D)-serine, R₄ is aresidue of (D)-alanine; and R₅ is a residue of N-methy-(L)-lleucine; or

when R₂ is so defined, X₂ is a linking group of 1-30 atoms, excludinghydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derviative of MeBmt, ora derivative of MeBmt in which the hydroxyl group is acylated with anacyl group of 1-10 atoms or a fluoresceinyl moiety; R₃ is a residue ofsarcosine, a residue of (D)-serine, or a residue of (D)-serine in whichthe hydroxyl is acylated with an acyl group of 1-10 atoms; R₄ is aresidue of (D)-alanine; and R₅ is a residue of (N)-methy-(L)-lleucine;or

when R₃ is so defined, X₁ is a linking group of 1-20 atoms, excludinghydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, ora derivative of MeBmt in which the R₂ is hydroxyl group is acylated withan acyl group of 1-20 atoms; R₄ is a residue of (D)-alanine; and R₅ is aresidue of (N)-methy-(N)-lleucine; or

where R₄ is so defined, X₁ is a linking group of 1-20 atoms, excludinghydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, ora derivative of MeBmt in which the hydroxyl group is acylated with anacyl group of 1-10 atoms; R₂ is a residue of (L)-alpha-aminobutyric acidor a residue of (L)-threonine, or a residue of (L)-threonine in whichthe hydroxyl group is acylated with an acyl group of 1-10 atoms; and R₅is a residue of (N)-methy-(N)-lleucine; or

where R₅ is so defined, X₁ is a linking group of 1-20 atoms, excludinghydrogen, R₁ is MeBmt, dihydro-MeBmt, a cyclized derivative of MeBmt, ora derivative of MeBmt in which the hydroxyl group is acylated with anacyl group of 1-10 atoms; R₂ is a residue of L-alpha-aminobutyric acidor a residue of (L)-threonine, or a residue of (L)-threonine in whichthe hydroxyl group is acylated with an acyl group of 1-10 atoms; and R₄is a residue of (D)-alanine;

and salts thereof.

Preferred cyclosporine derivatives of the present invention are of thestructures: ##STR11## where Fl is a fluorescent moiety; X₁ is a linkinggroup of 1-15 atoms, excluding hydrogen; R₁ is hydrogen, OH or OCOR₆ ;and R₆ is an alkyl group of from 1-6 carbon atoms or X₁ -Fl; and##STR12## where R₇ is hydrogen or an acyl group of 1-6 carbon atoms; R₈is hydrogen or CH₂ OR₇ ; X₂ is a linking group of 1-30 atoms, excludinghydrogen, and Fl is a fluorescent moiety; and ##STR13## where R₇ ishydrogen or an acyl group of 1-6 carbon atoms; R₉ is hydrogen or OR₇ ;X₁ is a linking group of 1-15 atoms, excluding hydrogen; and Fl is afluorescent moiety; and ##STR14## where R₇ is hydrogen or an acyl groupof 1-6 carbon atoms; X₁ is a linking group of 1-15 atoms, excludinghydrogen; and Fl is a fluorescent moiety; and ##STR15## where R₇ ishydrogen or an acyl group of 1-6 carbon atoms; X₁ is a linking group of1-15 atoms, excluding hydrogen; and Fl is a fluorescent moiety.

The present invention also provides a method and test kit employing suchcyclosporine tracer compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates general synthetic pathways for preparing cyclosporinetracer compounds according to the present invention.

FIG. 2 illustrates a calibration curve employed to determine the amountcyclosporine from a serum sample in a fluorescent polarizationimmunoassay using the cyclosporine tracer compounds of the presentinvention.

FIG. 3 illustrates a calibration curve employed to determine the amountcyclosporine from a whole blood sample sample in a fluorescentpolarization immunoassay using the cyclosporine tracer compounds of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The cyclosporine tracer compounds of the present invention are preparedaccording to the general reaction schemes set forth in FIG. 1., whereinR is a cyclosporine moiety, X is a linking group from between 1 and 6atoms, excluding hydrogen, Y is Cl or OCH₃, Z is a detectable moiety,and R' and R" are alkyl groups or functionalized alkyl groups as arecommonly found in carbodiimides.

For example, according to reaction scheme (i) of FIG. 1, cyclosporine orderivative thereof containing a free hydroxyl group is treated with asolution of phosgene in benzene or toluene to form an intermediatechoroformate. Alternatively, a similar intermediate can be formed usingcarbonyldiimidazole. The chloroformate is then reacted with, forexample, a fluorescein moiety which is substituted with an amino groupto form a carbamate linkage, as described in greater detail in examples3, 4, 5, 13, 14, 16, 17, 18 and 19 hereunder.

According to reaction scheme (ii) of FIG. 1, cyclosporine or aderivative thereof containing a free hydroxyl group is treated withoxalyl chloride to form the intermediate chlorooxalyl ester. Thisintermediate is then treated with, for example, a fluorescein moietywhich is substituted with an amino group to form an amide linkage, asdescribed in greater detail in examples 1 and 2 hereunder.

According to reaction scheme (iii),of FIG. 1, cyclosporine or aderivative thereof containing a free hydroxyl group or a free aminogroup is treated with succinic anhydride to form the intermediate acidhalf-ester or acid half-amide. The free carboxylic acid thus formed isthen activated employing carbodiimide, and subsequently treated with,for example, a fluorescein moiety substituted with an amino group, toform an amide linkage, and, alternatively, may proceed through theintermediacy of an active ester, such as an N-hydroxysuccinimide activeester, as described in greater detail in examples 11, 20, 21, 22, 24,25, 28, 29, 33, 34, 36, 38, 40 and 43 hereunder.

According to reaction scheme (iv) of FIG. 1, cyclosporine or aderivative thereof containing a free amino group is treated with acarboxyfluorescein active ester in the presence of a base. Thecyclosporine derivative and the fluorescein moiety are thus linkedthrough an amide bond, as described in greater detail in examples 11,12, 26 and 32 hereunder.

According to reaction scheme (v) of FIG. 1, cyclosporine or a derivativethereof containing a free amino group is treated with a fluoresceinmoiety substituted with a dichlorotriazinyl group to form anitrogen-carbon bond linkage, as described in greater detail in examples9, 10, 23 and 27 hereunder.

The detectable moiety component of the cyclosporine tracer compounds ofthe present invention can be selected from a variety of detectablelabels known in the art including, but not intended to be limited to,chemiluminescent molecules, luminescent molecules, enzymes, and thelike. According to the present invention, luminescent molecules known asfluorescein, and fluorescein derivatives, are preferred. Suchfluorescein derivatives include, but are not intended to be limited to,fluoresceinamine, carboxyfluorescein, (alpha-iodoacetamidofluorescein,4'-aminomethylfluorescein, 4'-N-alkylaminomethylfluorescein,5-aminomethylfluorescein, 6-aminomethylfluorescein,2,4-dichloro-1,3,5-triazin-2-yl-aminofluorescein (DTAF),4-chloro-6-methoxy-1,3,5-triazln-2-yl-aminofluorescein, andfluoresceinisothiocyanate. Particularly preferred derivatives are theaminomethylfluoresceins, the carboxyfluoresceins, and thefluoresceinamines.

Fluorescein exists in two tautomeric forms, depending on the acidconcentration (pH) of the environment. In the open (acid) form,fluorescein or a fluorescein derivative (or a tracer containing afluorescent molecule) is capable of absorbing blue light and emittinggreen fluorescence after an excited state lifetime of about fournanoseconds. When the open and closed forms coexist, relativeconcentration of molecules in the open and closed forms is easilyaltered by adjustment of the pH level. Generally, the cyclosporinetracer compounds of the present invention are prepared in solution asbiologically acceptable salts such as sodium, potassium, ammonium andthe like, which allows the compounds to exist in the fluorescent form.The specific salt present will depend on the buffer used to adjust thepH level. For example, in the presence of sodium phosphate buffer, thecompounds of the present invention will generally exist in the openform, as a sodium salt. Accordingly, the term "fluorescein" as usedherein, either as an individual compound or as a component of a tracer,is meant to include both the open and closed tautomeric forms, if theyexist for a particular molecule, except in the context of fluorescence,in which case an open form is necessary for the fluorescence to occur.As would be understood by one skilled in the art, fluorescent labels areideally chosen in accordance with their size, that is, the smaller themolecule, the more rapidly it will be able to rotate, and thus the moreeffective it will be as a fluorescence polarization immunoassay tracercompound. Such compounds provide fluorescent response when excited bypolarized light of an appropriate wavelength and thereby enable thefluorescence polarization measurement.

The cyclosporine tracer compounds according to the present invention canbe used to determine the presence or amount of cyclosporine, ormetabolites of cyclosporine, in diluted or undiluted test samples suchas whole blood, serum, plasma, spinal fluid, and the like, employingconventional immunoassay techniques known in the art. According to themethod of the present invention, a test sample suspected of containingcyclosporine, or cyclosporine and meteabolites of cyclosporine, iscombined with a cyclosporine tracer compound of the present inventionand an appropriate antibody thereto prepared according to methods knownin the art. Cyclosporine present in the test sample and the tracercompound compete for a limited number of binding sites on the antibody,resulting in the formation of cyclosporine-antibody and tracercompound-antibody complexes. By maintaining a constant concentration ofthe tracer compound and the antibody, the ratio of the formation ofcyclosporine-antibody complex to tracer-antibody complex is directlyproportional to the amount of cyclosporine in the test sample.

It is to be understood that the tracer compound of the present inventioncan be employed in immunoassay systems employing antibodies whichrecognize cyclosporine, or antibodies which recognize cyclosporine andmetabolites of cyclosporine. Monoclonal and polyclonal antibodies tocyclosporine have been described Donatsch, et al., supra; Quesniaux, etal., supra, and Quesniaux, et al., International Journal of Peptide andProtein Research, Vol. 31, pages 173-185 (1988); European PatentApplication Publication No. 283,801; Cacalano, et al., Journal ofImmunological Methods, Vol. 118(2), pages 257-263 (1989); andInternational Patent Application Publication No. WO 86/02080!.Accordingly, reference to the determination of cyclosporine as describedherein is intended to include the specific determination ofcyclosporine, independent from any metabolites which may be present in atest sample, or to the determination of cyclosporine and metabolitesthereof, which determination will of course depend upon the particularantibody employed in the immunoassay system, as described above.

As would be understood by one skilled in the art, the specificity of anantibody is determined, in part, by the structure of the immunogen usedto raise the antibody. Immunogens for small molecular weight analytesare prepared according to methods known in the art by coupling theanalyte to a large molecular weight carrier, such as a protein, througha covalent bond in order to ensure an adequate immune response in thelaboratory animal. The position of attachment of the carrier to theanalyte is such that recognition of the antibody for that site isgenerally low.

When preparing the tracer compound of the present invention, theposition of attachment of the detectable moiety to the derivatizedcyclosporine molecule, and the length and character of the linker armthat joins them, should be optimized such that there is a competitionbetween the tracer compound and cyclosporine from the test sample forbinding to the antibody. In many instances, it is advantageous to attachthe detectable moiety to a site on the cyclosporine molecule that is notwell recognized by the antibody, so that the antibody will neverthelessbind to the tracer compound. Typically, there may be sites other thanthe immunogen attachment site that are poorly recognized by theantibody. Accordingly, changing the length of the linker arm and thecharacter of the linker arm will often optimize the binding of antibodyto the tracer tracer compound to achieve the desired results.Furthermore, to be useful for the monitoring of cyclosporine, thecompetition between analyte and the tracer compound must be such thattherapeutic range levels can be distinguished from one another.

The structure of the tracer compound is important to the performance ofan immunoassay, and should be optimized for use with the antibodyemployed in the particular assay. For example, if the antibody binds tothe tracer compound with a high affinity, the tracer compound may not bedisplaced from the antibody by the analyte, or the tracer compoundcompetetively displaces all of the analyte from the antibody, whereinmeasurement of the analyte cannot be accomplished. Conversely, if theantibody does not recognize the tracer compound, no signal other thanany background signal can be detected and no measurement of analyte canbe accomplished. Similarly, the structure of the tracer compounddetermines, to some degree, the cross-reactivity of the antibody tometabolites or analogs of the analyte, since the relative bindingproperties of antibody with the analyte, analogs of the analyte, and thetracer compound determines the cross-reactivity.

The cyclosporine tracer compounds of the present invention arepreferably employed in fluorescence polarization immunoassay systemswherein the amount of cyclosporine in the test sample is determined byexciting the mixture with polarized light and measuring the polarizationof the fluorescence emitted by any of the free or unbound tracercompound and tracer-antibody complex. Any of the tracer compound whichis not complexed to an antibody is free to rotate in less than the timerequired for adsorption and re-emission of fluorescent light. As aresult, the re-emitted light is relatively randomly oriented so that thefluorescence polarization of any of the tracer compound not complexed tothe antibody is low, approaching zero. Upon complexing with a specificantibody, the tracer-antibody complex thus formed assumes the rotationof the antibody molecule, which is slower than that of the relativelysmall tracer compound molecule, thereby increasing the polarizationobserved. When making such determination, cyclosporine competes with thetracer compound for antibody sites wherein the observed polarization offluorescence of the tracer-antibody complex becomes a value between thevalue of the free tracer compound and the value tracer-antibody complex.Accordingly, if the test sample contains a high concentration ofcyclosporine or metabolites thereof, the observed polarization value iscloser to that of the free tracer compound, i.e., low. Conversely, ifthe test sample contains a low concentration of cyclosporine ormetabolites thereof, the polarization value is closer to that of thetracer-antibody complex, i.e., high. By sequentially exciting thereaction mixture of an immunoassay with vertically and then horizontallypolarized light, and analyzing only the vertical component of theemitted light, the polarization of the fluorescence in the reactionmixture can be accurately determined. The precise relationship betweenpolarization and concentration of cyclosporine is established bymeasuring the polarization values of calibrators having knownconcentrations, and the concentration of cyclosporine can beinterpolated from a standard curve prepared therefrom.

When employing fluorescence polarization techniques, the results can bequantified in terms of "millipolarization units", "span" (inmillipolarization units) and "relative intensity". The measurement ofmillipolarization units indicates the maximum polarization when amaximum amount of the tracer compound is bound to the antibody in theabsence of any analyte in the test sample. The higher the netmillipolarization units, the better the binding of the tracer compoundto the antibody. For the purposes of the present invention, a netmillipolarization value of at least about 130 is preferred.

The "span" is an indication of the difference between the netmillipolarization and the minimum amount of the tracer compound bound tothe antibody. A larger span provides for a better numerical analysis ofthe data. For the purposes of the present invention, a span of at leastabout 15 millipolarization units is preferred.

The "relative intensity " is a measure of the strength of thefluorescence signal above the background fluorescence. Thus, a higherintensity will give a more accurate measurement. The intensity isdetermined as the sum of the vertically polarized intensity plus twicethe horizontally polarized intensity. The intensity can range from asignal of about three times to about thirty times the background noise,depending upon the concentration of the tracer compound and other assayvariables. For the purpose of the present invention, an intensity ofabout three to about twenty times that of background noise is preferred.

When performing an immunoassay method employing a tracer compoundaccording to the present invention, the pH can be from between about 4.0to about 9.0, preferably from between about 6.0 and 8.0, most preferablyfrom between about 7.0 and 7.5. Where the detectable moiety of thetracer compound is a fluorescein moiety, the pH of the immunoassaysystem in which such tracer compound is employed must be sufficient toallow the fluorescein moiety of the tracer compound to exist in the openform. Various buffers can be used to achieve and maintain the pH duringan immunoassay procedure and include, but are not intended to be limitedto borate, phosphate, carbonate, Tris™, barbital and the like. Althoughany of such buffers can be employed, Tris and phosphate buffers arepreferred when performing a fluorescent polarization immunoassay.

The method according to the present invention is carried out at moderatetemperatures, preferably at a constant temperature. The temperature willnormally be from between about 0° C. to about 50° C., preferably fromabout 15° C. to about 40° C.

As will be described in greater detail hereinafter, the cyclosporinetracer compounds of the present invention have been found to beparticularly useful in a fluoresence polarization immunoassay whereinfrom between about 10⁻⁶ M to about 10⁻¹⁰ M of cyclosporine in a testsample can be determined. As would be understood by one skilled in theart, higher concentrations of cyclosporine can be determined by dilutingthe test sample. Although the concentration range of cyclosporine in atest sample will determine the range of concentration of the assayreagents such as the tracer compound and the antibody, the respectivereagent concentrations can be determined empirically to optimize thesensitivity of the assay, as can be determined by one of ordinary skillin the art.

According to a preferred embodiment of the present invention, thereagents for performing a fluorescent polarization immunoassay include afluorescent tracer compound comprising 4-aminomethylfluorescein coupledto the hydroxyl group of MeBmt at the first position of cyclosporine, asdescribed in Example 4 hereunder and represented by the formula##STR16## where R₁ is hydrogen, X₁ is a ##STR17## moiety, and Fl isfluorescein coupled at the 4' position thereof, and a monoclonalantibody to cyclosporine, such as described by International PatentApplication Publication No. WO 86/02080. The use of such fluorescenttracer compound of the formula was found to be surprisingly useful withsuch monoclonal antibody since such antibody was prepared with animmunogen coupled to a carrier protein molecule through the amino acidat the second position of cyclosporine, wherein the binding propertiesof a cyclosporine antibody are otherwise particularly sensitive tostructural changes at the first position. As will described in greaterdetail in the Examples hereunder, a cyclosporine monoclonal whole bloodprecipitation solution comprising methanol, ethylene glycol and zincsulfate as described in the copending U.S. patent application Ser. No.07/567,853, entitled "Protein Precipitation Reagent", filed on Aug. 15,1990, now U.S. Pat. No. 5,135,875issued Aug. 4, 1992 and incorporated byreference herein, and a solubilization reagent comprising saponin and adetergent such as Tergitol™ alkyloxy(polyethyleneoxy)propyleneoxyisoproponol!, such as described in the copending U.S. patentapplication Ser. No. 07/567,840, entitled "Solubilization Reagent ForBiological Test Samples", filed on Aug. 15, 1990, now abandoned, andincorporated by reference herein, are also employed. In addition, adilution buffer, calibrators and controls are preferably employed.

A preferred immunoassay procedure according to the present invention isa homogeneous immunoassay wherein the fluorescence polarization readingsare taken from a solution containing antibody-fluorescent tracercompound complexes and free or unbound fluorescent tracer compounds, andtherefore not requiring separation of such species. Such immunoassayprocedure is particularly advantageous over, for example,radioimmunoassay procedures where the bound radioactive tracer must beseparated from the unbound radioactive tracer before a reading can betaken.

According to the preferred assay procedure of the present invention, thetest sample containing cyclosporine, or cyclosporine and metabolitesthereof, are combined with the precipitation reagent described above,mixed and centrifuged, wherein a pellet of denatured protein isobtained. It is to be understood that cyclosporine and metabolites ofcyclosporine have a particularly high binding affinity for proteins,especially lipoproteins. Accordingly, in order to separate cyclosporineand metabolites of cyclosporine from such proteins which would otherwiseinterfere with the immunoassay determination of cyclosporine andmetabolites thereof as provided herein, the precipitation reagent isemployed to accomplish such separation wherein proteins present in atest sample are precipitated while, at the same time, recovering frombetween about 90% and 110% of the cyclosporine or cyclosporine andcyclosporine metabolites present in the test sample. Similarly, wherethe test sample is, for example, a whole blood test sample or otherbiological test sample containing various cellular components, it isdesirable to dissociate any cyclosporine or cyclosporine and metabolitesthereof from such cellular components in order to render anycyclosporine and metabolites thereof available for binding to theantibody. Accordingly, the solubilization reagent described above isemployed to dissociate any cyclosporine or cyclosporine and metabolitesthereof from such cellular components of the test sample.

Once the interfering proteins have been precipitated as described aboveand, in the case of, for example, a whole blood test sample, the samplefirst treated with the solubilization reagent as described above, thesupernatant containing cyclosporine, or cyclosporine and metabolites ofcyclosporine, is then combined with the antibody. Prior to addition ofthe tracer compound and dilution buffer, a background fluorescencereading is taken, wherein after an incubation period of from betweenabout ten minutes and about thirty minutes, a fluorescence polarizationreading is taken as described above.

A test kit according to the present invention comprises all of theessential reagents required to perform a desired immunoassay accordingto the present invention. The test kit is presented in a commerciallypackaged form as a combination of one or more containers holding thenecessary reagents, as a composition or admixture where thecompatibility of the reagents will allow. Particularly preferred is atest kit for the fluorescent polarization immunoassay determination ofcyclosporine, or cyclosporine and metabolites of cyclosporine,comprising an appropriate fluorescent tracer compound of the presentinvention, an appropriate antibody reagent, a precipitation reagent and,where the test sample is a whole blood test sample, a solubilizationreagent as described above. It is to be understood that the test kitcan, of course, include other materials as are known in the art andwhich may be desirable from a commercial user standpoint, such asbuffers, diluents, standards, and the like.

The present invention will now be illustrated, but is not intended to belimited, by the following examples:

EXAMPLE 1

O-(Chlorooxalyl)MeBmt!¹ cyclosporine

Cyclosporine (34.3 mg, 0.0285 mmoles) and dimethylaminopyridine (30.2mg, 0.247 mmoles) were dissolved in oxalyl chloride (1.0 mL) at 0° C.The flask was fitted with a stirbar and a drying tube, and the reactionwas stirred on an ice bath for 3.5 hours. The reaction was concentratedto dryness in vacuo. The residue was taken up into 1.0 mL of drydimethylformamide to make a 0.03M solution, and used in subsequentreactions.

EXAMPLE 2

O-(Fluorescein-5-ylaminooxalyl)MeBmt!¹ cyclosporine

The DMF solution described in Example 1 (0.33 mL, 9.5 μmoles) wascombined with fluoresceinamine isomer I (5.2 mg, 15 μmoles) in astoppered flask fitted with a stirbar. Pyridine was added until theapparent pH (determined by spotting the solution on moist pH paper) wasapproximately 4-5. The reaction stirred at room temperature for 3 days.The solvent was removed in vacuo and the residue taken up in 0.5 mL ofmethanol and applied to a 0.5 mm silica gel plate (20×20 cm). The platewas developed in 15% methanol/methylene chloride. The fluorescent bandat Rf 0.5 was removed from the silica gel with methanol and repurifiedon a 0.5 mm silica gel plate (20×20 cm), eluting twice with 5%methanol/methylene chloride. The desired band (Rf 0.37) was removed fromthe silica gel with-methanol.

EXAMPLE 3

O-(Chloroformyl)MeBmt-!¹ cyclosporine (Cyclosporine chloroformate)

Cyclosporine (24.2 mg, 0.020 mmoles) was dissolved in a 25% w/w solutionof phosgene in benzene (2.0 mL) in a 10 mL round bottom flask fittedwith stopper and stirbar. The reaction was stirred for 5 minutes todissolve the cyclosporine, then was allowed to stand undisturbed at roomtemperature for 24 hours. The reaction was concentrated in vacuo, andthe product could be stored as a solid at 0° C. for up to six months.For subsequent reactions, a 0.02M solution in DMF was used.

EXAMPLE 4

O-(Fluorescein-4'-ylmethylaminoformyl)MeBmt!¹ cyclosporine

Cyclosporine chloroformate, as a 0.02M solution in DMF as described inExample 3 (0.2 mL, 4 μmoles) was combined with 4-aminomethylfluoresceinhydrochloride (2.0 mg, 5 moles) in a stoppered vial fitted with astirbar. Pyridine was added until the apparent pH (by moist pH paper)was approximately 7. The reaction was stirred at room temperature for 24hours. The solvent was removed in vacuo, and the residue was taken up inmethanol and loaded onto a 1 mm silica gel plate. The plate wasdeveloped with 15% methanol/methylene chloride. The product band, Rf0.55, was eluted from the silica gel with methanol.

EXAMPLE 5

O-(Fluorescein-5-ylmethylaminoformyl)MeBmt!¹ cyclosporine

Cyclosporine chloroformate (5 mg of the solid described in Example 3,4.0 umole) was dissolved in 150 uL of dry DMF in a stoppered vial fittedwith a stirbar. 5-Aminomethylfluorescein hydrochloride (3.2 mg, 8 umole)and triethylamine (2.2 uL, 16 umole) were added, and the reactionstirred at room temperature for 2.5 days. The solvent was removed invacuo, and the residue was taken up in methanol and applied to a 1 mmsilica gel plate, which was eluted with 15% methanol/methylene chloride.The fluorescent band at Rf 0.64 was isolated and removed from the silicagel with methanol.

EXAMPLE 6

O-(Chloroacetyl)MeBmt!¹ cyclosporine

Cyclosporine (1.01 g, 0.840 mmoles) was dissolved in chloroacetylchloride (3.0 mL) in a round bottom flask fitted with stirbar and dryingtube. Dimethylaminopyridine (152.4 mg, 1.25 mmoles) was added. Thereaction was stirred at room temperature for 2.5 days. The reactionsolution was poured into 10 mL of cold (0°) saturated NaHCO₃ andstirred, while solid NaHCO₃ was added portionwise until bubbling ceased,about 2 hours. The solution was extracted with 3×20 mL of diethyl ether.The combined ethereal extracts were washed with 1×10 mL of 0.1N HCl,3×10 mL of water, and 1×10 mL of saturated NaCl solution. The organiclayer was dried over anhydrous MgSO₄, filtered, and concentrated invacuo to give 1.02 g of a yellow glassy residue. This material wassubjected to flash chromatography on 75 g of silica gel using 5%methanol/methylene chloride as eluent. Fractions containing product werecombined and concentrated to give 0.52 g of the title compound (48%yield). Fast-atom bombardment mass spectrometry showed and (M+H) signalat 1278.

EXAMPLE 7

O-(Azidoacetyl)MeBmt!¹ cyclosporine

O-(Chloroacetyl)MeBmt!¹ cyclosporine (103.3 mg, 0.0808 mmoles) andsodium azide (6.5 mg, 0.10 mmoles) were combined in a round bottom flaskfitted with stirbar and reflux condenser. Dimethylformamide (1.0 mL) and1 drop of water (to dissolve the sodium azide) were added. The reactionwas stirred at 50° C. overnight, then 90° C. for 1.5 hours. The solutionwas taken up into 20 mL of ether and washed with 3×10 mL of water and1×5 mL of saturated NaCl solution, dried over MgSO₄, filtered andconcentrated in vacuo to give 92.1 mg (88%) of a slightly yellow solid.TLC (silica gel, 5% methanol/methylene chloride) shower minorimpurities. IR showed azide absorption at 2100 cm-1.

EXAMPLE 8

O-(Glycyl)MeBmt!¹ cyclosporine

O-(Azidoacetyl)MeBmt!¹ cyclosporine (46.1 mg, 0.0359 mmoles) wasdissolved in 5.0 mL of absolute ethanol in a 100 mL Parr hydrogenationbottle. 5% Palladium on calcium carbonate, poisoned with lead (36.1 mg,78% w/w), and triethylamine (100 uL) were added, and the reaction wasshaken on a Parr apparatus at 50 psi H2 at room temperature overnight.The reaction was removed from the apparatus and filtered through a padof Celite. The Celite was washed with additional ethanol. The combinedfiltrate and washings were concentrated in vacuo to give 42 mg of amixture of two components by TLC (silica gel, 5% methanol/methylenechloride, Rf's 0.3 and 0.18). The mixture was separated on aChromatotron (Harrison Research, 810 Moana Court, Palo Alto, Calif.)using a 1 mm rotor and eluting with 5% methanol/methylene chloride.Fractions containing pure product were combined to give 23.4mg ofO-(glycyl)cyclosporine, 52% yield. FAB MS showed (M+H)+1259 and(M+Na)+1271 for the desired compound.

EXAMPLE 9

O-(5-Fluorescein-5-ylamino-3-chlorotriazinylglycyl)MeBmt!¹ cyclosporine

O-(Gycyl)MeBmt!1cyclosporine (Example 8, 5 mg, 4 umole) was dissolved in8 uL of methanol in a stoppered vial equipped with a stirbar.3,5-Dichlorotriazinylaminofluorescein isomer I (DTAF-I, 4.0 mg, 8 umole)was added, and the reaction was stirred at room temperature for 3.5days. The solution was loaded onto a 1 mm silica gel plate and developedwith 20% methanol/methylene chloride. The band at Rf 0.75 was elutedfrom the silica gel with methanol and repurified on a 1 mm silica gelplate, developing with 5% methanol/methylene chloride. The band at Rf0.3 was eluted from the silica gel with methanol.

EXAMPLE 10

O-(5-Fluorescein-6-ylamino -3-chlorotriazinylglycyl)MeBmt!¹ cyclosporine

The procedure in Example 9 was followed, usingdichlorotriazinylaminofluorescein isomer II (DTAF-II) in the place ofDTAF-I. The reaction was stirred for 1 day. The first purification wasdone with 20% methanol/methylene chloride (Rf 0.70) and the secondpurification was done with 10% methanol/methylene chloride (Rf 0.71).

EXAMPLE 11

O-(Fluorescein-5-carboxylglycyl)MeBmt!¹ cyclosporine

O-(Glycyl)MeBmt!¹ cyclosporine (Example 8, 5 mg, 4 umole) and theN-hydroxysuccinimide ester of 5-carboxyfluorescein (3.0 mg, 8 umole)were combined in a stoppered vial fitted with a stirbar, withdimethylformamide (50 uL), triethylamine (3.3 uL, 24 umole), anddimethylaminopyridine (5 umole). The reaction was stirred at roomtemperature overnight. The volatiles were removed in vacuo, and theresidue was taken up in methanol and loaded onto a 1 mm silica gelplate. The plate was developed with 20% methanol/methylene chloride, andthe band at Rf 0.64 was removed from the silica gel with methanol.Repurification by preparative thin layer chromatography with 2×10%methanol/methylene chloride gave a single band, Rf 0.43.

EXAMPLE 12

O-(Fluorescein-6-carboxylglycyl)MeBmt!¹ cyclosporine

The procedure in Example 11 was followed using the N-hydroxysuccinimideester of 6-carboxyfluorescein. The Rf of the desired band after 1development with 20% methanol/methylene chloride was 0.65; after thesecond purification with 2×10% methanol/methylene chloride the Rf was0.4.

EXAMPLE 13

O-(N-fluorescein-4'-ylmethyl acetamidoaminoformyl)MeBmt!¹ cyclosporine

Cyclosporine chloroformate, as a solution in DMF (Example 3, 4 moles)was combined with 4'-N-glycylaminomethylfluorescein hydrochloride (2.4mg, 5.3 μmoles) in a stoppered vial fitted with a stirbar. Pyridine(approx. 10 drops) was added until the apparent pH was about 8. Thereaction was stirred at room temperature for 1 day. The volatiles wereremoved in vacuo and the residue was taken up into methanol and loadedonto a 1 mm silica gel plate. The plate was eluted with 15%methanol/methylene chloride. The band at Rf 0.5 was eluted from thesilica gel with methanol. Repurification using 20% methanol/methylenechloride gave a band at Rf 0.6.

EXAMPLE 14

O-(Fluorescein-5-ylaminoformyl)MeBmt!¹ cyclosporine

Cyclosporine chloroformate, as a solution in DMF (Example 3, 4 moles)was combined with fluoresceinamine isomer I (6.2 mg, 18 μmoles) in astoppered vial fitted with a stirbar. Pyridine was added until theapparent pH was about 7. The reaction was stirred at room temperaturefor 1 day. The volatiles were removed in vacuo and the residue was takenup into methanol and loaded onto a 1 mm silica gel plate. The plate waseluted with 15% methanol/methylene chloride. The band at Rf 0.57 waseluted from the silica gel with methanol. Repurification using 10%methanol/methylene chloride gave a band at Rf 0.5.

EXAMPLE 15

O-AcetylThr!² cyclosporine

Thr!² cyclosporine (cyclosporin C, obtained from Sandoz AG, Basle,Switzerland; 0.30 g, 0.25 mmole) was dissolved in dry pyridine (1.0 mL)in a round bottom flask fitted with stirbar and drying tube. Thesolution was cooled to 0° C. on an ice bath. Acetic anhydride (28 uL,0.30 mmole) was added, and the ice bath was removed. After stirring atroom temperature for 3 hours, more acetic anhydride (28 uL, 0.60 mmolestotal) was added. The reaction was stirred at room temperatureovernight, and another 10 uL of acetic anhydride was added (total 0.7mmole). After another 6 hours stirring at room temperature, the reactionwas taken up into 25 mL of ether and washed with 1.2N HCl (25 mL), water(25 mL) and saturated NaCl solution (25 mL). The organic layer was driedover anhydrous MgSO₄, filtered and concentrated in vacuo. The residuewas taken up in CH₂ Cl₂ and cyclohexane to remove traces of acetic acid.The title compound was obtained in 82% yield (259 mg). Structure of theproduct was confirmed by 200 MHz NMR, which revealed the disappearanceof a triplet at delta 4.15 and appearance of a doublet at delta 5.6 anda singlet at delta 1.9.

EXAMPLE 16

O-ChlorocarbonylMeBmt!¹ O-acetylThr! ² cyclosporine

O-AcetylThr!² cyclosporine (Example 15, 17.3 mg, 13.7 mole) wasdissolved in a 25% w/w solution of phosgene in benzene (1.0 mL) in around bottom flask fitted with a tight stopper and stirbar. Afterstirring for 5 minutes to completely dissolve the peptide, the reactionstood at room temperature overnight. The volatiles were removed in vacuoto leave an off-white solid residue.

EXAMPLE 17

O-(Fluorescein-4'-ylmethylaminoformyl)Me Bmt!¹ O-acetylThr!²cyclosporine

O-ChlorocarbonylMeBmt!¹ O-acetylThr!² cyclosporine (Example 16, 4.6μmoles) in 0.3 mL of dry pyridine, was combined with4'-aminomethylfluorescein hydrochloride (5.5 mg, 13.8 μmoles) in astoppered vial fitted with a stirbar. The reaction was stirred at roomtemperature for 3 days. The solvent was removed in vacuo, and theresidue was taken up in methanol and loaded onto a 1 mm silica gelplate. The plate was developed with 15% methanol/methylene chloride. Theproduct band, Rf 0.95, was eluted from the silica gel with methanol.Repurification using 2×5% methanol/methylene chloride gave the productas a single band at Rf 0.4. The band was removed from the silica gelwith methanol.

EXAMPLE 18

O-(Imidazol-1-ylcarbonyl)Thr!² cyclosporine

Thr!² cyclosporine (14.6 mg, 12.0 moles) was placed in a round bottomflask fitted with stirbar and drying tube. Carbonyldiimidazole (14.8μmoles), dimethylaminopyridine (14.5 μmoles), dimethylformamide (44 μL)and methylene chloride (120 μL) were added. The reaction was stirred atroom temperature for 24 hours. The volatiles were removed, and theresidue was carried on immediately to subsequent reactions.

EXAMPLE 19

O-(Fluorescein-4'-ylmethylaminocarbonyl)Thr!² cyclosporine

O-(Imidazol-1-ylcarbonyl)Thr!² cyclosporine (Example 18, 6 moles) and4'-aminomethylfluorescein hydrochloride (5.2 mg, 13 moles) were combinedin 100 L of dry dimethylformamide in a stoppered vial fitted with astirbar. 4-Methylmorpholine (3 μL, 27 moles) was added, and the reactionwas stirred at room temperature overnight. The volatiles were removedand the residue was taken up into methanol. The solution was purified ontwo 0.5 mm silica gel plates, which were eluted with 15%methanol/methylene chloride. The bands at Rf 0.9 were eluted from thesilica gel with methanol to isolate the title compound.

EXAMPLE 20

O-(N-(N-(Fluorescein-4'-ylmethyl)carboxamidomethyl)carboxamidomethyl)Thr!.sup.2cyclosporine

(a) O-(Succinimid-N-yloxycarbonylmethyl)Thr!² cyclosporine

O-(Carboxymethyl)Thr!² cyclosporine (obtained from Sandoz AG, Basle,Switzerland; 20.3 mg, 15.9 μmoles) and N-hydroxysuccinimide (7.8 mg,67.8 μmoles) were dissolved in dry dimethylformamide (400 μL) in a roundbottom flask fitted with a stirbar and drying tube. 1-Ethyl-1'-(3'-dimethylamino)propyl!carbodiimide hydrochloride (9.2 mg, 48 μmoles)was added, and the reaction was stirred at room temperature overnight.The reaction solution was used in subsequent reactions withoutpurification.

(b)O-(N-(N-(Fluorescein-4'-ylmethy)carboxamidomethyl)carboxamidomethyl)Thr!.sup.2cyclosporine

O-(Succinimid-N-yloxycarbonylmethyl)Thr!² cyclosporine (as a reactionsolution, Example 20, part (a), 4 μmoles) andN-glycyl-4'-aminomethylfluorescein hydrochloride (6.2 μmoles) werecombined in a stoppered vial equipped with a stirbar. 4-Methylmorpholine(2.0 L, 18.2 μmoles) was added. The reaction stirred at room temperatureovernight. The volatiles were removed under high vacuum. The residue wastaken up into methanol and applied to a 0.5 mm silica gel plate, whichwas eluted with 15% methanol/methylene chloride. The band at Rf 0.9 waseluted from the silica gel with methanol and repurified, eluting with 5%methanol/methylene chloride. The band at Rf 0.45 was removed from thesilica gel with methanol.

EXAMPLE 21

O-(N-(Fluorescein-4'-ylmethyl)carboxamidomethyl)Thr!² cyclosporine

O-(Succinimid-N-yloxycarbonylmethyl)Thr!² cyclosporine (as a reactionsolution, Example 20, 4 μmoles) and 4'-aminomethylfluoresceinhydrochloride (3.1 mg, 7.8 moles) were combined in a stoppered vialfitted with a stirbar. 4-Methylmorpholine (2.0 L, 18.2 moles) was added.The reaction stirred at room temperature overnight. The volatiles wereremoved under high vacuum. The residue was taken up into methanol andapplied to a 0.5 mm silica gel plate, which was eluted with 15%methanol/methylene chloride. The band at Rf 0.8 was eluted from thesilica gel with methanol and repurified in the same manner, eluting 5%methanol/methylene chloride. The band at Rf 0.55 was removed from thesilica gel with methanol.

EXAMPLE 22

O-(N-methyl-N-(fluorescein-4-ylmethyl)carboxamidomethyl)Thr!²cyclosporine

O-(Succinimid-N-yloxycarbonylmethyl)Thr!² cyclosporine (as a reactionsolution, Example 20, 4 μmoles) and 4'-methylaminomethylfluoresceinhydrochloride (2.8 mg, 6.8 μmoles) were combined in a stoppered vialfitted with a stirbar. 4-Methylmorpholine (2.0 L, 18.2 μmoles) wasadded. The reaction stirred at room temperature overnight. The volatileswere removed under high vacuum. The residue was taken up into methanoland applied to a 0.5 mm silica gel plate, which was eluted with 15%methanol/methylene chloride. The band at Rf 0.92 was eluted the silicagel with methanol and repurified in the same manner, eluting with 2×5%methanol/methylene-chloride. The band at Rf 0.50 was removed from thesilica gel with methanol.

EXAMPLE 23

O-(5-Fluorescein-5-ylamino-3-chlorotriazinyl-2-aminoethyl)Thr!²cyclosporine

O-(2-aminoethyl)Thr!² cyclosporine (obtained from Sandoz AG, Basle,Switzerland; 5 mg, 4 moles) and DTAF-I (4 mg, 8 μmoles) were dissolvedin 60 L of methanol in a stoppered vial equipped with a stirbar, andstirred at room temperature overnight. The solution was applied to a 0.5mm silica gel plate and eluted with 20% methanol/methylene chloride. Theband at Rf 0.46 was removed from the silica gel with methanol, andrepurified in the same manner using 2×5% methanol/methylene chloride.The band at Rf 0.5 was removed from the silica gel with methanol.

EXAMPLE 24

O-(Fluorescein-4'-ylmethylaminosuccinyl)Thr!² cyclosporine

O-(Succinimid-N-yloxysuccinyl)Thr!² cyclosporine (obtained from SandozAG, Basle, Switzerland; 5 mg, 3.5 μmoles), 4'-aminomethylfluoresceinhydrochloride (2.9 mg, 7 μmoles), dimethylaminopyridine (6.5 μmoles),and triethylamine (1 μL, 7 moles)were combined in 65 μL of drydimethylformamide. The reaction was stirred at room temperature for 2.5days. The solvent was removed in vacuo, the residue was taken up inmethanol and applied to a 1 mm silica gel plate. The plate was elutedwith 10% methanol/methylene chloride and the band at Rf 0.8 was elutedfrom the silica gel with methanol. The band was repurified in the samemanner, using 2×5% methanol/methylene chloride and 1×10%methanol/methylene chloride to develop the plate. The band at Rf 0.7 wasremoved from the silica gel with methanol.

EXAMPLE 25

O-(N-(Fluorescein-4'-ylmethyl)carboxamidomethylaminosuccinyl)Thr!²cyclosporine

O-(Succinimidyloxysuccinyl)Thr!² cyclosporine (obtained from Sandoz AG,Basle, Switzerland; 5 mg, 3.5 μmoles), 4'-glycylaminomethylfluoresceinhydrochloride (2.9 mg, 7 μmoles), dimethylaminopyridine (3.5 μmoles),and triethylamine (1 L, 7 μmoles)were combined in 100 μL of drydimethylformamide. The reaction was stirred at room temperature for 2.5days. The solvent was removed in vacuo, the residue was taken up inmethanol and applied to a 1 mm silica gel plate. The plate was elutedwith 20% methanol/methylene chloride and the band at Rf 0.85 was elutedfrom the silica gel with methanol. The band was repurified in the samemanner, using 1×10% methanol/methylene chloride and 1×20%methanol/methylene chloride to develop the plate. The band at Rf 0.75was removed from the silica gel with methanol.

EXAMPLE 26

O-(N-(Fluorescein-6-ylcarbonylaminoethyl)aminosuccinyl)Thr!²cyclosporine

(a) O-(N-(2-BOC-aminoethyl)aminosuccinyl)Thr!² cyclosporineO-(Succinimidyloxysuccinyl)Thr!² cyclosporine (15 mg, 10.7 μmoles),mono-BOC-ethylenediamine (4.6 mg, 28.7 μmoles), anddimethylaminopyridine (2 μmoles) were combined in 150 L of drydimethylformamide. The reaction was stirred at room temperatureovernight. The reaction was taken up in 25 mL of ether and washed with4×10 ml of water and 1×10 mL of saturated NaCl solution. The organiclayer was dried over anhydrous sodium sulfate, filtered andconcentrated. After subjecting to high vacuum overnight, the residue waspurified on a Chromatotron using a 1 mm rotor and eluting with 5%-10%methanol/methylene chloride. Pure fractions were combined andconcentrated to give 14.7 mg (10 μmoles, 93% yield) of the titlecompound. FAB MS shows (M+H)⁺ 1460.

(b) O-(N-(2-Aminoethyl)aminosuccinyl)Thr!² cyclosporine

O-(N-(2-BOC-aminoethyl)aminosuccinyl)Thr!² cyclosporine (Example 26;14.7 mg, 10 μmoles) was dissolved in trifluoroacetic acid (300 μL) at 0°C. and stirred at that temperature overnight. The reaction was pouredonto 0.5 g of NaHCO₃ and 10 g of ice. After bubbling ceased, thesolution was extracted with 3×20 mL of methylene chloride. The organicextracts were combined and dried over anhydrous sodium sulfate.Filtration and concentration gave 10.5 mg of product.

(c) O-(2-Fluorescein-6-ylcarbonylaminoethyl)aminosuccinyl)Thr!²cyclosporine

O-(N-(2-Aminoethyl)aminosuccinyl)Thr!² cyclosporine and6-(succinimidooxycarbonyl)fluorescein (Research Organics; 2.3 mg, 4.9μmoles) were combined in dry dimethylformamide (100 μL) in a stopperedvial equipped with a stirbar. 4-Methylmorpholine (1 drop) was added togive an apparent pH of 7-8. The reaction was stirred at room temperatureovernight. The volatiles were removed in vacuo and the residue was takenup in methanol and applied to 2-0.5 mm silica gel plates. The plateswere developed with 2×15% methanol/methylene chloride. The band at Rf0.8 was eluted from the silica gel with methanol to give the titlecompound.

EXAMPLE 27

O-(2-(3-Chloro-5-(fluorescein-5-ylamino)triazin-1-yl)aminoethylaminosuccinyl)Thr!²cyclosporine

O-(N-(2-Aminoethyl)aminosuccinyl)Thr!² cyclosporine (3.5 mg, 2 μmoles)and dichlorotriazinylaminofluorescein isomer I (DTAF-I, 2.5 mg, 5μmoles) were combined in 100 L of methanol in a stoppered vial fittedwith a stirbar. The reaction was stirred at room temperature overnight,then applied to a 0.5 mm silica gel plate and eluted with 2×15%methanol/methylene chloride. The band at Rf 0.5 was removed from thesilica gel with methanol to give the title compound.

EXAMPLE 28

O-(N-(Fluorescein-4'-ylmethyl)-N-methyl-aminosuccinylpoly(oxyethyl)succinyl)Thr!²cyclosporine

a. O-(Poly(oxyethyl)succinyl)Thr!² cyclosporine

O-(Succinyl)Thr!² cyclosporine (350 mg, 0.265 mmole) was dissolved indry CH₂ Cl₂ (0.5 mL) with polyethylene glycol (avg. MW 200 g/mole, 82.2mg, approx. 0.91 mmole) N-ethyl-N'-dimethylaminopropylcarbodiimidehydrochloride (90.2 mg, 0.471 mmole), and dimethylaminopyridine (46.7mg, 0.382 mmole), in a round bottom flask fitted with stirbar and dryingtube. The reaction stirred at room temperature overnight. The solutionwas taken up in 20 mL of ether and washed with 2×5 mL of 0.12N HCl, 2×10mL of water, 2×5 mL of 5% NaHCO₃, 3×10 mL of water, and 1×5 mL ofsaturated NaCl solution. The organic layer was dried over anhydrous Na₂SO₄, filtered and concentrated to give 299.4 mg of a white foam. Theproduct was purified on a Chromatotron using a 1 mm rotor and elutingwith 3% methanol/methylene chloride. Fractions containing pure productwere combined and concentrated to give 240.6 mg (60% yield). FAB MS gavefour parent peaks, differing in mass by 44: (M+H)⁺ 1538, 1494, 1450,1406.

b. O-(Hydroxysuccinylpoly(oxyethyl)succinyl)Thr!² cyclosporine

O-(Poly(oxyethyl)succinyl)threonyl!² cyclosporine (Example 28; 83.8 mg,55.9 μmoles), succinic anhydride (8.9 mg, 89 μmoles), anddimethylaminopyridine (80 moles) were dissolved in DMF (1 mL) in a roundbottom flask fitted with stirbar and drying tube. The reaction wasstirred at room temperature overnight. The solution was taken up intoether (25 mL) and washed with 5mL of 0.12N HCl, 3×5 mL of water,and 10mL of saturated NaCl solution. The organic layer was dried overanhydrous MgSO₄, filtered and concentrated to give 67.4 mg (86%) of thetitle compound.

c. O-(N-(Fluorescein-4'-ylmethyl)-N-methylaminosuccinylpoly(oxyethyl)succinyl)Thr!² cyclosporine

O-(Succinylpoly(oxyethyl)succinyl)Thr!² cyclosporine (Example 28; 5 mg,3.1 moles), diisopropylcarbodiimide (0.58 L, 3.7 μmoles),N-hydroxybenzotriazole (1.1 mg, 7 μmoles), triethylamine (1.4 L, 10μmoles),and 4'-methylaminomethylfluorescein (1.4 mg, 3.7 μmoles) werecombined in DMF (150 L) in a stoppered vial fitted with a stirbar. Thereaction was stirred at room temperature overnight. The volatiles wereremoved in vacuo, and the residue was taken up in methanol and appliedto a 0.5 mm silica gel plate. The plate was eluted with 20%methanol/methylene chloride. The desired compound was removed from theband at Rf 0.8 with methanol.

EXAMPLE 29

DihydroMeBmt!1 O-(fluorescein-4-ylmethylaminosuccinyl)Thr!² cyclosporine

(a) DihydroMeBmt!¹ O-(succinyl)Thr!² cyclosporine

Dihydrocyclosporine C was reacted with succinic anhydride according tothe procedure to give the title compound. (b) DihydroMeBmt!¹O-(N-fluorescein-4'-ylmethylaminosuccinyl)Thr!² cyclosporine.

DihydroMeBmt!¹ O-(succinyl)Thr!² cyclosporine (0.8 mg, 0.6 μmole) wascombined with diisopropylcarbodiimide (0.21 L, 1.3 μmoles),N-hydroxybenzotriazole (0.34 mg, 2.2 μmoles), 4'-aminomethylfluoresceinhydrochloride (0.54 mg, 1.3 μmoles), and triethylamine (0.45 L, 3.3μmoles) in DMF (50 L) in a stoppered vial equipped with a stirbar. Thereaction was stirred at room temperature overnight. The volatiles wereremoved in vacuo, and the residue was taken up into methanol and appliedto a 0.5 mm silica gel plate. The plate was eluted with 20%methanol/methylene chloride. The band at Rf 0.61 was collected and thecompound removed from the silica gel with methanol. Repurification inthe same manner using 2×15% methanol/methylene chloride gave the productband at Rf 0.5. The compound was removed from the silica gel withmethanol to give the title compound.

EXAMPLE 30

O-(Fluorescein-4'-ylmethylaminocarbonyl)-(D)-MeSer!³ cyclosporine(D)-MeSer!³ cyclosporine (13.3 mg, 10.8 μmoles),1,1'-carbonyldiimidazole (13 moles), and dimethylaminopyridine (13μmoles) were combined in DMF (0.5 mL) in a round bottom flask fittedwith stirbar and drying tube. The reaction stirred at room temperaturefor 24 hours. To one-third of this reaction mixture was added4'-aminomethylfluorescein hydrochloride (3.6 mg, 9 μmoles) and4-methylmorpholine (2.0 L, 18.2 μmoles). The apparent pH was 8-9 bymoist pH paper. The reaction was further stirred at room temperature for24 hours. The volatiles were removed in vacuo, and the residue was takenup into methanol and applied to a 1 mm silica gel plate. The plate waseluted with 15% methanol/methylene chloride. The band at Rf 0.6 wasremoved, the compound was eluted from the plate with methanol, and theproduct was repurified in the same manner, eluting with 2×10%methanol/methylene chloride. The band at Rf 0.72 was collected, and thetitle compound was isolated by removal from the silica gel withmethanol.

EXAMPLE 31

Thr!² O-(4'-Fluorescein-4'-ylmethylaminocarbonyl)-(D)-MeSer!³cyclosporine

Thr!² (D)-MeSer!³ cyclosporine(28.5 mg, 22.8 μmoles),1,1'-carbonyldiimidazole (3.3 mg, 20 μmoles), and dimethylaminopyridine(13 μmoles) were combined in DMF (150 L) in a round bottom flask fittedwith stirbar and drying tube. The reaction stirred at room temperaturefor 24 hours. To one-fifth of this reaction mixture was added4'-aminomethylfluorescein hydrochloride (4.9 mg, 12.3 μmoles) and4-methylmorpholine (1 drop). The apparent pH was 8-9 by moist pH paper.The reaction was further stirred at room temperature for 24 hours. Thevolatiles were removed in vacuo, and the residue was taken up intomethanol and applied to a 1 mm silica gel plate. The plate was elutedwith 15% methanol/methylene chloride. The band at Rf 0.57 was collected,and the title compound was isolated by removal from the silica gel withmethanol.

EXAMPLE 32

(Fluorescein-5-ylcarbonyl)amino Ala!⁸ cyclosporine

AminoAla!⁸ cyclosporine (obtained from Sandoz AG, Basle, Switzerland;3.0 mg, 2.5 μmoles), 5-(succinimidooxycarbonyl)fluorescein (ResearchOrganics; 3.1 mg, 6.5 moles), and 4-methylmorpholine (2.0 L, 18 moles)were combined in DMF (100 L) in a stoppered vial equipped with stirbar.The reaction was stirred at room temperature for 2.5 days. The volatileswere removed in vacuo, and the residue was taken up into methanol andapplied to a 0.5 mm silica gel plate. The plate was eluted with 15%methanol/methylene chloride. The band at Rf 0.45 was collected, and thetitle compound was isolated by removal from the silica gel withmethanol.

EXAMPLE 33

epsilon-(N-(Fluorescein-4'-ylmethyl)carboxamidomethylamino-succinyl)-(D)-Lys!⁸cyclosporine

N-(Succinimidooxysuccinyl)-(D)-Lys!⁸ cyclosporine (obtained from SandozAG, Basle, Switzerland; 3.0 mg, 2.1 μmoles) and4'-glycylaminomethylfluorescein (1.8 mg, 4.2 moles) were combined in DMF(50 L) with dimethylaminopyridine (5 μmoles) and triethylamine (8.4moles) in a stoppered vial equipped with a stirbar. The reaction stirredovernight at room temperature. The volatiles were removed in vacuo, andthe residue was taken up into methanol and applied to a 0.5 mm silicagel plate. The plate was eluted with 20% methanol/methylene chloride.The band at Rf 0.70 was collected, and the title compound was isolatedby removal from the silica gel with methanol.

EXAMPLE 34

O-(N-(Fluorescein-4'-ylmethyl)-N-propylaminosuccinyl)MeThr!¹⁰cyclosporine

MeThr!¹⁰ cyclosporine (obtained from Sandoz AG, Basle, Switzerland; 20mg, 17 moles), succinic anhydride (27.3 mg,0.273 mmoles), anddimethylaminopyridine (11.1 mg, 0.091 mmoles) were combined in pyridine(250 L) in a stoppered vial equipped with a stirbar. The reaction wasstirred at 45° C. for 3 days. The reaction was taken up into 10 mL ofether and washed with 10 mL of 1N HCl. The aqueous extracts wereback-extracted with 5 mL of ether. The combined organic extracts werewashed with 5 mL of water and 5 mL of saturated NaCl solution, thendried over anhydrous MgSO4, filtered and concentrated to give 16 mg ofO(-succinyl)MeThr!¹⁰ cyclosporine, slightly contaminated with thestarting material and the bis-succinyl derivative.(O-Succinyl)MeThr!10cyclosporine (4 mg, 3.1 μmoles) was combined withdicyclohexylcarbodiimide (6 moles), N-hydroxybenzotriazole (6 moles),4'-aminomethylfluorescein hydrochloride (12 moles), and triethylamine(3.1 μmoles) in DMF (100 L) in a stoppered vial equipped with a stirbar.The reaction was stirred at room temperature overnight. The volatileswere removed in vacuo, and the residue was taken up into methanol andapplied to a 0.5 mm silica gel plate. The plate was eluted with 15%methanol/methylene chloride. The band at Rf 0.50 was collected andrepurified in the same manner with 2×10% methanol/methylene chloride.The band at Rf 0.2 was collected and the title compound was isolated byremoval from the silica gel with methanol.

EXAMPLE 35

O-AcetylMeBmt!¹ O-succinylThr!² cyclosporine

O-SuccinylThr!² cyclosporine (20.8 mg, 15.8 μmoles) anddimethylaminopyridine (32.5 mg, 0.266 mmole were combined in aceticanhydride (0.5 mL, 5.3 mmoles) in a round bottom flask fitted withstopper and stirbar. The reaction stirred at room temperature for 3days. The solution was poured into 0° C. 5% NaHCO3 with stirring, andsolid NaHCO₃ was added portionwise until the pH of the solution wasabout 5. The aqueous solution was extracted with 3 portions of ethylacetate. The combined organic layers were washed with water andsaturated NaCl solution, then dried over anhydrous MgSO₄. Filtration andconcentration gave a residue which was concentrated again from CH₂ Cl₂and cyclohexane to remove traces of acetic acid. The product waspurified on a Chromatotron using a 1 mm rotor and 5-10%methanol/methylene chloride. Fractions containing pure product werecombined and concentrated to give 12.6 mg (9.3 μmoles, 58% yield) of thetitle compound.

EXAMPLE 36

O-AcetylMeBmt!¹ O-(fluorescein-4-ylmethylaminosuccinyl)Thr!²cyclosporine

O-AcetylMeBmt!¹ O-succinylthreonyl!² cyclosporine (5 mg, 3.7 μmoles) wascombined with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (1.4 mg, 7.3 μmoles) and N-hydroxysuccinimide (1.0 mg, 8.7μmoles) in 37 μL of methylene chloride and stirred at room temperatureovernight. The reaction was taken up into 5 mL of CH₂ Cl₂ and washedwith 5 mL of water. The water layer was back-extracted with 5 mL of CH₂Cl₂, and the combined organic layers were washed with saturated NaClsolution (5 mL) and dried over anhydrous MgSO₄, filtered, andconcentrated in vacuo. Triethylamine (1.0 L, 7.4 μmoles),dimethylaminopyridine (5 μmoles), and aminomethylfluoresceinhydrochloride (2.3 mg, 5.5 μmoles) in DMF (87 L) were added, and thereaction stirred overnight. The volatiles were removed in vacuo, and theresidue was taken up into methanol and applied to a 0.5 mm silica gelplate. The plate was eluted with 20% methanol/methylene chloride. Theband at Rf 0.83 was collected and the compound removed from the silicagel with methanol. Repurification in the same manner using 1×10%methanol/methylene chloride gave the product band at Rf 0.74. Thecompound was removed from the silica gel with methanol to give the titlecompound.

EXAMPLE 37

O-(Chloroacetyl)MeBmt!¹ O-succinylThr!² cyclosporine

O-SuccinylThr!² cyclosporine (100.7 mg, 76.4 μmoles) anddimethylaminopyridine (23.1 mg, 0.19 mmole) were combined inchloroacetyl chloride (1.0 mL) in a round bottom flask fitted withdrying tube and stirbar. The reaction stirred at 45° C. overnight. Thesolution was cooled to room temperature and the volatiles were removedon high vacuum. The residue was taken up into 1 mL of acetone andtreated with 1 mL of 1M NaOAc solution at 0° C. for 1 hour to hydrolyzethe acid chloride. The solution was extracted with 20 mL of ethylacetate. The organic layer was washed with water (2×5 mL) and saturatedNaCl solution (5 mL), then dried over anhydrous MgSO₄. Filtration andconcentration gave 105.4 mg. The product was purified on a Chromatotronusing a 1 mm rotor and 4.5 methanol/0.5% acetic acid/95% methylenechloride. Fractions containing pure product were combined andconcentrated to give 53.9 mg (38.6 moles, 51% yield) of the titlecompound.

EXAMPLE 38

O-(Chloroacetyl)MeBmt!¹ O-(fluorescein-4'-ylmethylaminosuccinyl)Thr!²cyclosporine

O-(Chloroacetyl)MeBmt!¹ O-succinylThr!² cyclosporine (11.3 mg, 8.1μmoles), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(3.4 mg, 17.7 μmoles) and N-hydroxysuccinimide (3.2 mg, 27.8 μmoles),and 4-methylmorpholine (5 L, 45.5 μmoles) were combined in DMF (1.0 mL)in a round bottom flask fitted with stirbar and drying tube. Thereaction was stirred at room temperature overnight. One-third of thissolution was combined with 4'-aminomethylfluorescein hydrochloride (2.2mg, 5.3 μmoles). Additional 4-methylmorpholine was added to bring theapparent pH to 8-9. The reaction was stirred at room temperatureovernight. The volatiles were removed in vacuo, and the residue wastaken up into methanol and applied to a 0.5 mm silica gel plate. Theplate was eluted with 15% methanol/methylene chloride. The band at Rf0.9 was collected and the compound removed from the silica gel withmethanol to give the title compound.

EXAMPLE 39

O-(Azidoacetyl)MeBmt!¹ O-succinylThr!² cyclosporine

The procedure of Example 7 was followed, using O-(chloroacetyl)MeBmt!¹O-succinylThr!² cyclosporine (27.0 mg, 19.3 μmoles), sodium azide (12.7mg, 0.192 mmoles), and 1.0 mL of DMF. Yield 19.4 mg, 13.8 μmoles, 72%.

EXAMPLE 40

O-(Azidoacetyl)MeBmt!¹ O-(fluorescein-4'-ylmethylaminosuccinyl)Thr!²cyclosporine

The procedure of Example 37 was followed, using O-(azidoacetyl)MeBmt!¹O-succinylThr!² cyclosporine (15.2 mg, 10.8 μmoles),1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride (5.8 mg,30.3 μmoles), N-hydroxysuccinimide (4.8 mg, 42 μmoles),4-methylmorpholine (4 L, 36.4 μmoles), and 4'-aminomethylfluoresceinhydrochloride 3.9 mg,9.8 μmoles). The band at Rf 0.87 was collected andthe compound removed from the silica gel with methanol. Repurificationin the same manner gave a band at Rf 0.85, which was eluted withmethanol to give the title compound.

EXAMPLE 41

(3-(R)-Methyl-5-allyl-2-tetrahydrofuranyl)sarcosyl!¹ Thr!² cyclosporine

Thr!² cyclosporine (50 mg, 41 μmoles) was dissolved in 0.7 mL of dry CH₂Cl₂ in a round bottom flask equipped with N² inlet and stirbar, andcooled to -78° C. with a dry ice/acetone bath. Phenylselenyl chloride(7.9 mg, 41 μmoles) was dissolved in CH₂ Cl₂ (80 μL) and added dropwiseto the cold peptide solution. The reaction was stirred for 25 minutes at-78° C. Meta-chloroperbenzoic acid (8.1 mg, 47 μmoles) was dissolved in80 L of CH₂ Cl₂ and added dropwise to the reaction. The dry ice/acetonebath was allowed to come to room temperature as the reaction stirred.After 4 hours the reaction was taken up into EtOAc (10 mL) and washedwith 5% NaCO3 (10 mL), 2×5 mL of water, and 5 mL of saturated NaClsolution. The organic layer was dried over anhydrous MgSO₄, filtered andconcentrated. The residue was purified on 25 g of flash-grade silicagel, using 5% methanol/methylene chloride to elute the column. Fractionscontaining pure product were combined and concentrated to give 36.7 mg(73.6% yield) of the title compound.

EXAMPLE 42

(3-(R)-Methyl-5-allyl-2-tetrahydrofuranyl)sarcosyl!¹ (O-succinyl) Thr!²cyclosporine

(3-(R)-Methyl-5-allyl-2-tetrahydrofuranyl)sarcosyl!¹ Thr!² cyclosporine(36.7 mg, 30.2 μmoles) was combined with succinic anhydride (3.3 mg, 33μmoles) and pyridine (4.6 L, 60.4 μmoles) in 100 L of DMF in a roundbottom flask fitted with stirbar and drying tube. The reaction stirredat room temperature for 4 days. The volatiles were removed in vacuo andthe residue was taken up in CH₂ Cl₂ (10 mL) and washed with water (5mL). The aqueous layer was back-extracted with 2×5 mL of CH₂ Cl₂. Thecombined organic layers were dried over anhydrous MgSO₄, filtered andconcentrated to 20.0 mg of the title compound.

EXAMPLE 43

(3-(R)-Methyl-5-allyl-2-tetrahydrofuranyl)sarcosyl!¹O-(fluorescein-4'-ylmethylaminosuccinyl)Thr)!² cyclosporine

(3-(R)-Methyl-5-allyl-2-tetrahydrofuranyl)sarcosyl!¹ O-succinyl Thr!²cyclosporine (10 mg, 7.6 moles), diisopropylcarbodiimide (1.3 L, 8.4μmoles), aminomethylfluorescein hydrochloride (3.3 mg, 8.4 μmoles),triethylamine (4.0 L, 28 μmoles) and dimethylaminopyridine (10 moles)were combined in DMF (100 L) in a round bottom flask equipped with astirbar and drying tube. The reaction was stirred at room temperatureovernight. The volatiles were removed in vacuo, and the residue wastaken up into methanol and applied to a 0.5 mm silica gel plate. Theplate was eluted with 20% methanol/methylene chloride. The band at Rf0.64 was collected and the compound removed from the silica gel withmethanol to give the title compound.

EXAMPLE 44

O-(Fluorescein-4'-ylmethylaminoformyl)MeBmt!¹ O-benzoylSer!³cyclosporine

Ser!³ cyclosporin; 20 mg, 16 moles) was dissolved in pyridine (200 L) ina stoppered vial fitted with stirbar. Benzoyl chloride (2.1 L, 18μmoles) and dimethylaminopyridine (5 mg, 41 μmoles) were added. Thereaction was stirred at 45° C. for 2 days. The volatiles were removed invacuo and the crude reaction mixture was treated as in Examples 3 and 4.The volatiles were removed in vacuo and the residue was taken up intomethanol and applied to a 1 mm silica gel plate, which was developedwith 1×15% methanol/methylene chloride. The fluorescent band at Rf 0.6was removed from the silica gel with methanol, and was repurified in thesame manner, developing the plate with 2×10% methanol/methylenechloride. The band at Rf 0.3 was removed from the silica gel withmethanol to give the title compound.

EXAMPLE 45

O-AcetylMeBmt!¹ O-(fluorescein-4'-ylmethyl)carboxymethylThr!²cyclosporine

O-(Carboxymethyl)Thr)!² cyclosporine (obtained from Sandoz AG, Basle,Switzerland 10 mg, 8 μmoles) was dissolved in 200 L of dry methylenechloride. Acetyl chloride (1.7 L, 24 μmoles) was added, and the reactionwas stirred at room temperature for 2.5 days. The reaction was taken upinto methylene chloride (1 mL), washed with 1N HCl (1 mL) and saturatedNaCl solution (1 mL), dried over anhydrous MgSO₄, filtered andconcentrated to give O-AcetylMeBmt!¹ O-(carboxymethyl)Thr!²cyclosporine. This was then treated as in Example 20, part (a), and asin Example 21. The volatiles were removed in vacuo and the residue wastaken up into methanol and applied to a 1 mm silica gel plate, which wasdeveloped with 1×15% methanol/methylene chloride. The fluorescent bandat Rf 0.6 was removed from the silica gel with methanol to give thetitle compound.

EXAMPLE 46

Cyclosporine Serum and Whole Blood Fluorescent PolarizationInmmunoassays.

Reagents

The reagents for performing a fluorescence polarization immunoassayaccording to the present invention were prepared as follows:

(a) Cyclosporine Tracer Reagent

A 60 nanomolar cyclosporine tracer reagent was prepared comprising thecyclosporine tracer compound prepared according to Example 4 in 0.1Msodium phosphate buffer, pH 7.5, containing 0.01 % (w/v) bovine gammaglobulin, 0.1% (w/v) soldium azide, 5.0% (w/v) ethylene glycol and 0.05%(w/v) Tween™ 20.

(b) Monoclonal Antibody Formulation

A monoclonal antibody reagent was prepared comprising mouse (ascites)monoclonal antibody to cyclosporine (Sandoz AG, Basle, Switzerland)diluted with a citrate buffer containing sodium azide.

(c) Pretreatment Reagent

A pretreatment reagent was prepared comprising 0.1M Tris™ buffer, pH7.5, 0.1% (w/v) sodium azide, 0.5% (w/v) copper sulfate and 10.0% (w/v)5-sulfosalicylate.

(d) Dilution Buffer

A dilution buffer was prepared comprising 0.1M sodium phosphate, pH 7.5,and 0.1% (w/v) bovine gamma globulin.

(e) Serum Precipitation Reagent

A serum precipitation reagent was prepared comprising 10 mM zinc sulfatein an aqueous diluent with 70% (w/v) ethylene-glycol, 25% (w/v)methanol, and 0.5 grams 5-sulfosalicylic acid.

(f) Whole Blood Precipitation Reagent

A whole blood precipitation reagent was prepared comprising 60 mM zincsulfate, 50& (w/v) methanol and 30% (w/v) ethylene glycol.

(g) Solubilization Reagent

A solubilization reagent was prepared comprising 2.0% (w/v) Tergitol minfoam™, 2.0% (w/v) saponin and 0.1% (w/v) sodium azide.

(h) Calibrators

(1) Cyclosporine monoclonal whole blood calibrators were preparedcomprising cyclosporine and an artificial human whole blood matrix. Thecalibrators were prepared at concentrations of 0.0, 100, 250, 500, 1000,and 1500 nanograms per milliliter, with sodium azide as a preservative.

(2) Cyclosporine monoclonal serum calibrators were prepared comprisingcyclosporine and a serum matrix. The calibrators were prepared atconcentrations of 0.0, 30, 60, 120, 240 and 400 nanograms per milliliterwith sodium azide as a preservative.

(i) Controls

(1) Cyclosporine monoclonal whole blood controls were preparedcomprising cyclosporine and an artificial human whole blood matrix. Thecontrols were prepared at concentrations of 150, 400 and 800 nanogramsper milliliter, with 0.1% sodium azide as a preservative.

(2) Cyclosporine monoclonal serum controls were prepared comprisingcyclosporine and a serum matrix. The controls were prepared atconcentrations of 45, 90 and 320 nanograms per milliliter with sodiumazide as a preservative.

Cyclosporine Serum FPIA Assay Protocol

A fluorescent polarization immunoassay for determining cyclosporine in aserum sample employing an Abbott TDx^(R) Therapeutic Drug MonitoringAnalyzer was performed as follows:

Fifty microliters each of patient serum samples containing cyclosporine,controls and calibrators were pipetted into labeled centrifuge tubes. Apipette was filled with the serum precipitation reagent, purged of airbubbles, and 150 microliters were dispensed into each centrifuge tube bytouching the end of the pipette tip to the wall of each centrifuge tubewhile dispensing the reagent. The centrifuge tubes were then capped andmixed on a vortex mixer for ten seconds and placed into a centrifugehead so that the tubes were evenly distributed so that the centrifugehead was balanced. The tubes were centrifuged for approximately threeminutes at 9,500×g until a clear supernatant and a hard, compact pelletof denatured protein was obtained. After centrifugation was complete,each tube was uncapped and the supernatant was decanted into thecorresponding sample well of a TDx Sample Cartridge. Since 150microliters of supernatant were required to perform the assay inaccordance with the preferred TDx assay procedure, each centrifuge tubewas tapped on the edged of the corresponding sample well of the SampleCartridge in order to recover all of the supernatant.

The fluorescence polarization value of each calibrator, control andsample was determined and printed on the output tape of the Abbott TDxAnalyzer. A standard curve was generated in the instrument by plottingthe polarization, P, of each calibrator versus its concentration using anonlinear regression analysis wherein, the concentration of each controland sample was read off the stored calibration curve (FIG. 2) andprinted on the output tape.

The sensitivity of the preferred fluorescence polarization assayaccording to the present invention is 15.0 nanograms/milliliter ofcyclosporine and metabolites. When compared to an availableradioimmunoassay using 60 clinical samples, a linear least squaredregression analysis gave a slope of 0.947, an intercept of 7.15, and acorrelation coefficient of 0.969.

Where a test kit according to the present invention is being used inconjunction with the TDx Analyzer, the reagents for performing thefluorescent polarization immunoassay according to the present inventioncan be contained in separate vials of a TDx Reagent Pack wherein vialcaps from each of the vials in the Reagent Pack are removed and placedinto designated wells inside the Reagent Pack. Accordingly, once theReagent Pack is placed inside the TDx Analyzer, the assay procedureheretofore is fully automated.

If a manual assay is being performed, the test sample is first treatedwith the precipitation reagent as described above, and then mixed withthe dilution buffer. The antibody reagent and the pretreatment solutionare then placed into the test tube containing the sample, and abackground fluorescence reading is taken. The tracer compound anddilution buffer are added to the sample, and after incubation, afluorescence polarization reading is taken.

Cyclosporine Whole Blood FPIA Assay Protocol

A fluorescent polarization immunoassay for determining cyclosporine in awhole blood test sample employing an Abbott TDx^(R) Therapeutic DrugMonitoring Analyzer was performed as follows:

One hundred fifty microliters each of patient whole blood samplescontaining cyclosporine, controls and calibrators were pipetted intolabeled centrifuge tubes, and 50 microliters of the solubilizationreagent were added to each of the tubes. A pipette was filled with thewhole blood precipitation reagent, purged of air bubbles, and 300microliters were dispensed into each centrifuge tube by touching the endof the pipette tip to the wall of each centrifuge tube while dispensingthe reagent. The centrifuge tubes were then capped and mixed on a vortexmixer for ten seconds and placed into a centrifuge head so that thetubes were evenly distributed so that the centrifuge head was balanced.The tubes were centrifuged for approximately three minutes at 9,500×guntil a clear supernatant and a hard, compact pellet of denaturedprotein was obtained. After centrifugation was complete, each tube wasuncapped and the supernatant was decanted into the corresponding samplewell of a TDx Sample Cartridge and the fluorescence polarization valueof each calibrator, control and sample was determined and printed on theoutput tape of the Abbott TDx Analyzer as described above. A standardcurve was generated in the instrument by plotting the polarization, P,of each calibrator versus its concentration using a nonlinear regressionanalysis wherein, the concentration of each control and sample was readoff the stored calibration curve (FIG. 3) and printed on the outputtape.

It will be apparent that many modifications and variations of thepresent invention as herein set forth are possible without departingfrom the spirit and scope hereof, and that, accordingly, suchlimitations are imposed only as indicated by the appended claims.

We claim:
 1. A method for determining cyclosporine, or cyclosporine andmetabolites of cyclosporine, in a test sample, said method comprisingthe steps of:(a) contacting said test sample with a cyclosporinederivative corresponding to the formula: ##STR18## wherein ##STR19##represents a single or double bond; F1 is a detectable moiety from aluminescent molecule selected from the group consisting offluoresceinamines and carboxyfluoresceins;X1 is a linking group of 1-15atoms excluding hydrogen; R1 is hydrogen, OH or OCOR6; and R6 is analkyl group of from 1-6 atoms or X1-F1; and an antibody capable ofbinding to (i) cyclosporine, or cyclosporine and metabolites ofcyclosporine, and (ii) said cyclosporine derivative, to form a reactionsolution therewith, said cyclosporine derivative capable of producing adetectable fluorescence polarization response to the presence of saidantibody; (b) passing a plane of polarized light through the saidreaction solution to obtain a fluorescence polarization response; and(c) detecting said fluorescent polarization response to said reactionsolution as a function of the amount of cyclosporine, or cyclosporineand metabolites of cyclosporine, present in said test sample.
 2. Amethod for determining cyclosporine, or cyclosporine and metabolites ofcyclosporine, in a test sample, said method comprising the steps of:(a)contacting said test sample with a cyclosporine derivative correspondingto the formula: ##STR20## wherein ##STR21## represents a single ordouble bond; R7 is hydrogen or an acyl group of 1-6 carbon atoms;R8 ishydrogen or CH2OR7; X2 is a linking group of 1-30 atoms excludinghydrogen; and F1 is a detectable moiety from a luminescent moleculeselected from the group consisting of fluoresceinamines andcarboxyfluoresceins; and an antibody capable of binding to (i)cyclosporine, or cyclosporine and metabolites of cyclosporine, and (ii)said cyclosporine derivative, to form a reaction solution therewith,said cyclosporine derivative capable of producing a detectablefluorescence polarization response to the presence of said antibody; (b)passing a plane of polarized light through the said reaction solution toobtain a fluorescence polarization response; and (c) detecting saidfluorescent polarization response to said reaction solution as afunction of the amount of cyclosporine, or cyclosporine and metabolitesof cyclosporine, present in said test sample.
 3. A method fordetermining cyclosporine, or cyclosporine and metabolites ofcyclosporine, in a test sample, said method comprising the steps of:(a)contacting said test sample with a cyclosporine derivative correspondingto the formula: ##STR22## wherein ##STR23## represents a single ordouble bond; R7 is hydrogen or an acyl group of 1-6 carbon atoms;R9 ishydrogen or OR7; X1 is a linking group of 1-15 atoms excluding hydrogen;and F1 is a detectable moiety from a luminescent molecule selected fromthe group consisting of fluoresceinamines and carboxyfluoresceins; andan antibody capable of binding to (i) cyclosporine, or cyclosporine andmetabolites of cyclosporine, and (ii) said cyclosporine derivative, toform a reaction solution therewith, said cyclosporine derivative capableof producing a detectable fluorescence polarization response to thepresence of said antibody; (b) passing a plane of polarized lightthrough the said reaction solution to obtain a fluorescence polarizationresponse; and (c) detecting said fluorescent polarization response tosaid reaction solution as a function of the amount of cyclosporine, orcyclosporine and metabolites of cyclosporine, present in said testsample.
 4. A method for determining cyclosporine, or cyclosporine andmetabolites of cyclosporine, in a test sample, said method comprisingthe steps of:(a) contacting said test sample with a cyclosporinederivative corresponding to the formula: ##STR24## wherein ##STR25##represents a single or double bond; R7 is hydrogen or an acyl group of1-6 carbon atoms;X1 is a linking group of 1-15 carbon atoms excludinghydrogen; and F1 is a detectable moiety from a luminescent moleculeselected from the group consisting of fluoresceinamines andcarboxyfluoresceins; and an antibody capable of binding to (i)cyclosporine, or cyclosporine and metabolites of cyclosporine, and (ii)said cyclosporine derivative, to form a reaction solution therewith,said cyclosporine derivative capable of producing a detectablefluorescence polarization response to the presence of said antibody; (b)passing a plane of polarized light through the said reaction solution toobtain a fluorescence polarization response; and (c) detecting saidfluorescent polarization response to said reaction solution as afunction of the amount of cyclosporine, or cyclosporine and metabolitesof cyclosporine, present in said test sample.
 5. A method fordetermining cyclosporine, or cyclosporine and metabolites ofcyclosporine, in a test sample, said method comprising the steps of:(a)contacting said test sample with a cyclosporine derivative correspondingto the formula: ##STR26## wherein ##STR27## represents a single ordouble bond; R7 is hydrogen or an acyl group of 1-6 carbon atoms;X1 is alinking group of 1-15 atoms excluding hydrogen; and F1 is a detectablemoiety from a luminescent molecule selected from the group consisting offluoresceinamines and carboxyfluorescein; and an antibody capable ofbinding to (i) cyclosporine, or cyclosporine and metabolites ofcyclosporine, and (ii) said cyclosporine derivative, to form a reactionsolution therewith, said cyclosporine derivative capable of producing adetectable fluorescence polarization response to the presence of saidantibody; (b) passing a plane of polarized light through the saidreaction solution to obtain a fluorescence polarization response; and(c) detecting said fluorescent polarization response to said reactionsolution as a function of the amount of cyclosporine, or cyclosporineand metabolites of cyclosporine, present in said test sample.
 6. A testkit useful for the fluorescent polarization immunoassay determination ofthe amount of cyclosporine, or cyclosporine and metabolites ofcyclosporine, in a biological test sample, said test kit comprising:(a)a cyclosporine derivative corresponding to the formula: ##STR28##wherein ##STR29## represents a single or double bond; F1 is a detectablemoiety from a luminescent molecule selected from the group consisting offluoresceinamines and carboxyfluoresceins;X1 is a linking group of 1-15atoms excluding hydrogen; R1 is hydrogen, OH or OCOR6; and R6 is analkyl group of from 1-6 atoms or X1-F1; (b) an antibody capable ofbinding to (i) cyclosporine, or cyclosporine and metabolites ofcyclosporine, and (ii) said cyclosporine derivative, said cyclosporinederivative capable of producing a detectable fluorescence polarizationresponse to the presence of said antibody.
 7. The test kit of claim 6wherein said antibody is capable of binding to cyclosporine and saidcyclosporine derivative.
 8. The test kit of claim 6 wherein saidantibody is capable of binding to cyclosporine and metabolites ofcyclosporine and said cyclosporine derivative.
 9. The test kit of claim6, further comprising a precipitation reagent.
 10. The test kit of claim9, wherein said precipitation reagent comprises methanol, ethyleneglycol and zinc sulfate.
 11. The test kit of claim 6, further comprisinga solubilization reagent for whole blood test samples.
 12. The test kitof claim 11, wherein said solubilization reagent comprisesalkyloxy(polyethyleneoxypropyleneoxy)-isopropanol.
 13. The method ofclaim 1, wherein said antibody is capable of binding to cyclosporine andsaid cyclosporine derivative.
 14. The method of claim 1, furthercomprising the step of treating said test sample with a precipitationreagent.
 15. The method of claim 14, wherein said precipitation reagentcomprises methanol, ethylene glycol and zinc sulfate.
 16. The method ofclaim 1, wherein said test sample is whole blood.
 17. The method ofclaim 16, further comprising the step of treating said whole blood testsample with a solubilization reagent.
 18. The method of claim 17,wherein said solubilization reagent comprisesalkyloxy(polyethyleneoxypropyleneoxy)-isopropanol.